The present invention relates to high-speed parallel synthesis of combinatorial libraries and more particularly to a multifunctionalized solid support resin and to a method for the synthesis of combinatorial libraries using a multi-functional solid support resin.
The use of solid phase synthesis techniques for the synthesis of polypeptides and oligonucleotides are well known in the art. More recently, the use of solid phase techniques for the synthesis of small organic molecules has become a major focus of research. Of prime importance has been the ability of solid phase techniques to be automated, with an attendant increase in compound throughput and efficiency in research. This has been exploited with great vigor in the area of pharmaceutical research where it has been estimated that 10,000 compounds must be synthesized and tested in order to find one new drug (Science, 259, 1564, 1993). The focus on combinatorial chemistry techniques to increase compound throughput has now become almost universal in the pharmaceutical and agricultural industries.
An additional aspect relates to the chemical diversity of the compound stocks that are available for screening in pharmaceutical companies in the search for new lead structures. These have tended to be limited to the classes of compounds previously investigated through medicinal chemical techniques within each company. Therefore the availability of new classes of molecules for screening has become a major need.
Combinatorial chemistry involves both the synthesis and screening of large sets of compounds, called libraries. The libraries themselves can be arrays of individual compounds or mixtures. Therefore, the synthetic approaches are also classified into two categories, including combinatorial synthesis of mixtures and parallel synthesis leading to individual compounds. For screening purposes it is also important that the formed compounds be synthesized in 1 to 1 molar ratios.
In the first approach to creating molecular diversity, the combinatorial synthesis comprises multiple reactions in one reaction vessel resulting in the generation of all possible product combinations from a set of reactants. The simplest manifestation of the approach is to allow several reagents to react in solution at the same time to form all possible products. Among the examples is the synthesis of a library of over 97,000 members by reaction of a mixture of amines with 9,9-dimethylxanthene-2,4,5,7-tetracarboxylic acid tetrachloride (Carell, T; et al. Angew. Chem. Int. Ed. Engl. 33, 2059). However, this approach is usually unproductive unless the reagents are few and their reactivities are well matched to approach formation of the various compounds in 1 to 1 mol ratios.
Another approach is the use of the portioning-mixing method or the split synthesis (Furka, A; et al. Int. J. Pept. Protein Res. 37, 487, 1991). The synthesis is executed by repetition of three simple operations, including dividing a monofunctional solid support resin into equal portions, reacting each portion individually with one of the building blocks and then homogeneously mixing the portions. Starting with a single substance the number of compounds is tripled after each coupling step. For example, in the preparation of trimers, 27 different compounds can be prepared in three pools. These compounds can be cleaved into solution and screened as soluble pools, or the ligands can remain attached to the beads and screened in immobilized form. However, biological screens performed on such large mixtures of soluble compounds can be ambiguous since the observed activity could be due to a single compound or to a combination of compounds acting either collectively or synergistically. The subsequent identification of specific biologically active members is challenging, since the numbers of compounds present in the pools and their often limited concentration deter their isolation and re-assay. Because of this, the identification of individual active compounds from the library requires the repetitive re-synthesis and re-testing of the most active smaller subsets of the library until activity data are obtained on homogenous compounds. There is no direct method available to elucidate the chemical structures of large libraries of mixtures. However many methods have been developed to aid and accelerate the deconvolution process, including recursive deconvolution and multiple encoding approaches. There still remain a number of critical issues in screening libraries consisting of large mixtures of compounds.
By contrast, many other practitioners are using a method called parallel, or robotic, synthesis. This practice simply involves performing a series of individual reactions in separate vessels. Using traditional manual organic synthesis a chemist can synthesize only about 50 compounds per year. By the use of robots, which can perform multiple reactions simultaneously, this procedure can be made more efficient.
One of earliest examples of the parallel method for the synthesis of compounds is the multi-pin method developed by Geysen et al., for combinatorial solid-phase peptide synthesis (Geysen et al.; J. Immunol. Meth. (1987) 102:259-274). According to this method, a series of 96 pins are mounted on a block in an arrangement and spacing which correspond to a 96-well microtiter reaction plate, and the surface of each pin is derivatized to contain terminal linker functional groups. The pin block is then lowered into a series of reaction plates to immerse the pins in the wells of the plates where coupling occurs at the terminal linker functional groups, and a plurality of further reactions are carried out in a similar fashion. Reagents varying in their substituent groups occupy the wells of each plate in a predetermined array, to form a unique product on each pin. By using different combinations of substituents, one achieves a large number of different compounds with an array of central core structures.
Another type of solid phase parallel synthesis method is the diversomer approach from Park-Davis group (DeWitt, S. H.; et al. Proc. Natl. Acad. Sci. USA, 90, 6909, 1993). It was designed for the synthesis of small organic molecules. The solid support resin was placed into porous tubes immersed into tubes containing the various reagents which pass through the porous walls to contact the solid phase support resin.
A related method of synthesis uses porous polyethylene bags (Tea Bag method) containing the functionalized solid phase resins (Houghton, R. A., et al., Nature, 354, 84-86, 1991). These bags of resin can be moved from one reaction vessel to another in order to undergo a series of reaction steps for the synthesis of libraries of products.
As a consequence of the development of the efficient automation equipment and processes, the parallel synthesis technique has now become the most extensively used method in combinatorial chemistry. However, the libraries created using the parallel method (one compound per vessel) usually require more steps than those created using other combinatorial syntheses. As a result, more time is required to synthesize a comparable size library than would be required using other combinatorial techniques, such as the portioning-mixing method discussed above.
In view of the above, the field of pharmaceutical and agricultural research has a strong need for highly flexible technologies to generate a large number of novel classes of compounds for screening and clinical testing.
Solid Support Resins:
Solid support resin synthesis is carried out on a substrate consisting of a polymer, cross-linked polymer, functionalized polymeric pin, or other insoluble material. These polymers or insoluble materials have been described in literature and are known to those who are skilled in the art of solid phase synthesis (Stewart J M, Young J. D.; Solid Phase Peptide Synthesis, 2nd Ed; Pierce Chemical Company: Rockford. Ill., 1984). Some of them are based on polymeric organic substrates such as polyethylene, polystyrene, polypropylene, polyethylene glycol, polyacrylamide, and cellulose. Additional types of supports include composite structures such as grafted copolymers and polymeric substrates such as polyacrylamide supported within an inorganic matrix such as kieselguhr particles, silica gel, and controlled pore glass.
Examples of suitable support resins and linkers are given in various reviews (Barany, G.; Merrifield, R. B. xe2x80x9cSolid Phase Peptide Synthesis xe2x80x9d, in xe2x80x9cThe Peptidesxe2x80x94Analysis, Synthesis, Biologyxe2x80x9d. Vol 2, [Gross, E. and Meienhofer, J., Eds.], Academic Press, Inc., New York, 1979, pp 1-284; Backes, B. J.; Ellman, J. A. Curr. Opin. Chem. Biol. 1997. 1, 86; James, I. W., Tetrahedron 1999, 55, 4855-4946) and in commercial catalogs (Advanced ChemTech, Louisville, Ky.; Novabiochem, San Diego, Calif.). Some examples of particularly useful functionalized resin/linker combinations that are meant to be illustrative and not limiting in scope are shown below:
Aminomethyl Polystyrene Resin (Mitchell, A. R., et al., J. Org. Chem., 1978, 43, 2845): 
This resin is the core of a wide variety of synthesis resins. The amide linkage can be formed through the coupling of a carboxylic acid to amino group on solid support resin under standard peptide coupling conditions. The amide bond is usually stable under the cleavage conditions for most acid labile, photo labile and base labile or nucleophilic linkers.
Acid Labile Resins:
1. Wang resin (Wang, S. S.; J. Am. Chem. Soc. 1973, 95, 1328-1333). 
Wang resin is perhaps the most widely used of all resins for acid substrates bound to the solid support resin. The linkage between the substrate and the polystyrene core is through a 4-hydroxybenzyl alcohol moiety. The linker is bound to the resin through a phenyl ether linkage and the carboxylic acid substrate is usually bound to the linker through a benzyl ester linkage. The ester linkage has good stability to a variety of reaction conditions, but can be readily cleaved under acidic conditions, such as by using 25% TFA in DCM.
2. Rink resin (Rink, H.; Tetrahedron Lett. 1987, 28, 3787). 
Rink resin is used to prepare amides utilizing the Fmoc strategy. It has also found tremendous utility for a wide range of solid phase organic synthesis protocols. The substrate is assembled under basic or neutral conditions, then the product is cleaved under acidic conditions, such as 10% TFA in DCM.
3. Knorr resin (Bernatowicz, M. S., et al. Tetrahedron lett., 1989, 30, 4645). 
Knorr resin is very similar to Rink resin, except that the linker has been modified to be more stable to TFA. Typically, the product is cleaved from the Knorr resin using 95% TFA in DCM.
4. PAL resin (Bernatowicz, M. S., et al. Tetrahedron lett., 1989, 30, 4645). 
PAL resin is an acid labile resin developed for the synthesis of amides by Fmoc chemistry. Like Rink and Knorr resins, products are cleaved from PAL resin in the presence of TFA. However, compared to Knorr resin, PAL resin is more than two times as active towards cleavage.
5. HMBA-MBHA Resin (Sheppard, R. C., et al., Int. J. Peptide Protein Res. 1982, 20, 451). 
This resin has been widely used in solid phase organic synthesis and in peptide synthesis, especially in the synthesis of cyclic peptides, peptides containing C-terminal amino acid alcohols. The products can be cleaved from the resin using a variety of nucleophiles, such as hydroxides, amines or alkoxides to give carboxylic acids, amides and esters.
6. HMPA resin. This also is an acid labile resin which provides an alternative to Wang resin and represented as: 
7. Benzhydrylamine copoly(styrene-1 or 2%-divinylbenzene) which referred to as the BRA resin (Pietta, P. G., et al., J. Org. Chem. 1974, 39, 44). 
BHA resin is the first resin developed for preparing peptide C-terminal amides using Boc chemistry. The products can be cleaved under strong acidic conditions, such as using HF.
8. Methyl benzhydrylamine copoly(styrene-1 or 2%-divinylbenzene) which is referred to as MBHA and represented as: 
This resin can be cleaved more easily than benzhydrylamine resin. But strong acids are still required to effieciently cleave the products from the resin.
9. Trityl and functionalized Trityl resins, such as aminotrityl resin and amino-2-chlorotrityl resin (Barlos, K.; Gatos, D.; Papapholiu, G.; Schafer, W.; Wenqing, Y.; Tetrahedron Lett. 1989, 30, 3947). These are highly acid labile resin for the introduction of an amino group or for the synthesis of amides. 
10. Sieber amide resin (Sieber, P.; Tetrahedron Lett. 1987, 28, 2107). This resin is useful for preparing amides and amines. Like Rink resin, products can be cleaved using TFA in DCM. 
Because it is less sterically hindered than Rink resin, this resin allows for higher loading in more sterically demanding applications.
10. Rink acid resin (Rink, H., Tetrahedron Lett., 1987, 28, 3787). 
This resin can be cleaved under conditions as mild as 10% acetic acid. The nature of this resin is that the hydroxyl group can be considered weakly nucleophilic or electrophilic depending on the reaction conditions.
12. HMPB-BHA resin (4-hydroxymethyl-3-methoxyphenoxybutyric acid-BHA Florsheimer, A.; Riniker, B. in xe2x80x9cPeptides 1990; Proceedings of the 21st European Peptide Symposiumxe2x80x9d, [Giralt, E. and Andreu, D. Eds.], ESCOM, Leiden, 1991, pp 131. 
This is a super acid sensitive resin on which the carboxylic acids are released with 1% TFA in DCM.
Base Labile or Nucleophilic Resins:
1. Merrifield resinxe2x80x94Chloromethyl co-poly(styrene-1 or 2%-divinylbenzene) which can be represented as: 
A carboxylic acid substrate is attached to the resin through nucleophilic replacement of chloride under basic conditions. The resin is usually stable under acidic conditions, but the products can be cleaved under basic and nucleophilic conditions in the presence of amine, alcohol, thiol and H2O.
2. Hydroxymethyl polystyrene resin (Wang, S. S., J. Org. Chem., 1975, 40, 1235). 
The resin is an alternative to the corresponding Merrifield resin, whereas the substrate is attached to a halomethylated resin through nucleophilic displacement of halogen on the resin, the attachment to hydroxymethylated resins is achieved by coupling of activated carboxylic acids to the hydroxy group on the resin or through Mitsunobu reactions. The products can be cleaved from the resin using a variety of nucleophiles, such as hydroxides, amines or alkoxides to give carboxylic acids, amides and esters.
3. Oxime resin (DeGrado, W. F.; Kaiser, E. T.; J.Org. Chem. 1982, 47, 3258). 
This resin is compatible to Boc chemistry. The product can be cleaved under basic conditions.
Photolabile Resins (e.g. Abraham, N. A. et al.; Tetrahedron Lett. 1991, 32, 577):
The products can be cleaved from these resins photolytically under neutral or mild conditions, making these resins useful for preparing pH sensitive compounds. Examples of the photolabile resins include
1. ANP resin: 
2. alpha-bromo-alpha-methylphenacyl polystyrene resin: 
Safety Catch Resins (see resin reviews above; Backes, B. J.; Virgilio, A. A.; Eliman, J. Am. Chem. Soc. 1996, 118, 3055-6):
These resins are usually used in solid phase organic synthesis to prepare carboxylic acids and amides, which contain sulfonamide linkers stable to basic and nucleophilic reagents. Treating the resin with haloacetonitriles, diazomethane, or TMSCHN2 activates the linkers to attack, releasing the attached carboxylic acid as a free acid, an amide or an ester depending on whether the nucleophile is a hydroxide, amine, or alcohol, resepectively. Examples of the safty catch reasins include:
20. 4-sulfamylbenzoyl-4xe2x80x2-methylbenzhydrylamine resin: 
21. 4-sulfamylbutryl-4xe2x80x2-methylbenzhydrylamine resin: 
TentaGel Resins: 
TentaGel resins are polyoxyethyleneglycol (PEG) grafted (Tentagel) resins (Rapp, W.; Zhang, L.; Habich, R.; Bayer, E. in xe2x80x9cPeptides 1988; Proc. 20tth European Peptide Symposiumxe2x80x9d [Jung,G. and Bayer, E., Eds.], Walter de Gruyter, Berlin, 1989, pp 199-201. TentaGel resins, e.g. TentaGel S Br resin can swell in a wide variety of solvents and the bead size distribution is very narrow, making these resins ideal for solid phase organic synthesis of combinatorial libraies. TentaGel S Br resin can immobilize carboxylic acids by displacing the bromine with a carboxylic acid salt. The products can be released by saponification with dilute aqueous base. Resins with silicon linkage (Chenera, B.; Finkelstein, J. A.; Veber, D. F.; J. Am. Chem. Soc. 1995, 117, 11999-12000; Woolard, F. X.; Paetsch, J.; Ellman, J. A.; J. Org. Chem. 1997, 62, 6102-3). Some examples of these resins contain protiodetachable arylsilane linker and traceless silyl linker. The products can be released in the presence of fluoride. 
As described above, a wide variety of resins containing different linkers have been developed. The design of novel linkers suitable for the solid phase synthesis of small organic compounds has received great attention over the last few years. It is very clear that the quality of the combinatorial libraries synthesized on solid support resin, such as the range of yield and purity, depends not only on the synthetic strategy for the construction of the molecules but also on the nature of the resins and linkers to be chosen. The linkers can be divided into several classes based upon their stability, such as acid labile, base labile, photolabile, safety catch and traceless linkers. In conventional strategy for solid phase synthesis, the resin to be used contains the same linker so that the product is expected to be completely cleaved under an appropriate condition.
Recently, U.S. Pat. No. 5,635,598 disclosed selective cleavable linkers based on iminodiacetic acid ester and its application to solid phase peptide synthesis. The method is directed to cleavable linkers that can release peptide from the solid phase support resin under relatively mild conditions by formation of a diketopiperazine or other cyclic structure, such that the cyclic structure remains on the solid support resin, and, in a second cleavage, under more stringent conditions of high pH. This type of linker was claimed to be further combined with another cleavable linker which is also attached to the solid support resin backbone. The multiple linkers can then be cleaved selectively so that the desired peptide is sequentially released.
Another multiple release system based on a combination of benzyl ester type acid labile linkers with different sensitivities toward acid has been described (M. Cardno and M. Bradley, Tetrahedron lett., 37, 135, 1996. In this approach, three different linkers were coupled to the core resin, aminomethyl resin, in 1:1:1 molar ratio which was then applied to the peptide synthesis. The most acid labile linker releases the peptide product in 1% TFA, while the second linker needs 95% TFA. The third copy of the same peptide serves the purpose of analysis or on-bead assays. However, the synthesis of different products on these resins has not been reported yet.
A novel method based on the resin combination strategy was disclosed recently by us in a U.S. patent application Ser. No: 09/264,515 now abandoned. In this approach the multiple resins containing different linkers are combined in the same reaction vessel in which a plurality of chemical reactions are carried out to create multiple products on these solid supports respectively, which are then sequentially cleaved from the resins under the appropriate cleavage conditions. The method has been extensively used at Helios Pharmaceuticals in the synthesis of small organic combinatorial libraries.
Accordingly, it is an object of this invention to provide a single solid support resin that will provide for the production of a variety of different small organic compounds in a single reaction vessel.
Another object of this invention is to provide a flexible technology for high throughput parallel synthesis of combinatorial libraries.
Yet another object is to provide a method for efficiently forming combinatorial libraries in which the compounds are formed substantially in molar ratios of 1 to 1.
Yet another object of the invention is to provide a method for forming a variety of different compounds and for recovering the various compounds in a pure state without contamination by the other formed compounds.
The present invention is directed to multifunctionalized solid phase support resins for as the preparation of combinatorial libraries and to a method for high-speed parallel synthesis of combinatorial libraries utilizing multifuntionalized solid phase support resins. Such multifunctionalized support resins comprise polymers that contain a core template having multiple sites that can be attached by one or more linkers. The linkers incorporate reactive functionalities, (e.g. amino, hydroxyl, oximino, phenolic, silyl, carboxylic ester etc.) for loading of synthons, such as monomers, small molecules, oligomers and the like, suitable for carrying out a plurality of further reactions to synthesize the desired products. Each linker has a different functionality, one of them is chemically unstable under certain cleavage conditions (acid sensitive, base sensitive and the like) under which the functionality of the other linkers on the resin are inert. Accordingly, only the product at the unstable site is cleaved and individually separated from the reaction vessel while products at the stable site or sites remain attached to the support resin. The resin can be subsequently subjected to the further chemical manipulation for product synthesis or to subsequent cleavage steps under appropriate conditions so that a second product is cleaved and separated from the reaction vessel. Depending on the number of different linkers attached to the resin backbone, additional cleavage steps permit the sequential cleavage and separation of additional products. Therefore, products having different structures can be synthesized on a single solid support resin and can be released sequentially without cross contamination. The number of cleavage steps and cleavage conditions is dependent upon the number of different linker sites and the number of products to be released.
The invention further relates to a method for forming combinatorial libraries of compounds having different but related structures by solid phase methods. In accordance with the invention the method is carried out using a single multifunctionalized solid phase support resin and by sequentially separating and recovering the different products from the support resin.
The following abbreviations are used herein and unless specifically indicated otherwise, designate the following groups or chemical compounds.